Highly pathogenic influenza A viruses periodically infect both humans and nonhuman animals, including chickens. To gain insight into the origin of influenza outbreaks in poultry, we investigated two H5N2 viruses, A/chicken/Pennsylvania/13609/93 (Ck/PA/93) and A/chicken/Florida/25717/93 (Ck/FLA/93), that had been isolated in live-bird markets in Pennsylvania and Florida during surveillance studies in 1993. Phylogenetic analysis of the HA genes of these isolates, as well as H5N2 viruses isolated from ruddy turnstone surfbirds in 1991 (A/ruddy turnstone/Delaware/244/91 [RT/DE/91]) and other known H5 strains, indicated that Ck/PA/93 and Ck/FLA/93 share a common ancestor with RT/DE/91 and did not originate from A/chicken/Pennsylvania/1370/83 (Ck/PA/1370/83), which devastated chicken populations in 1983-1984. Both isolates were nonpathogenic in chickens by experimental infection and their HA protein (HA0) could not be cleaved into HA1 and HA2 without trypsin. The sequences at the HA cleavage sites of Ck/PA/93 and Ck/FLA/93 (R-K-T-R) appear to be intermediate between those of virulent and avirulent viruses, raising the possibility that a single mutation could promote virulence in chickens. We therefore recommend eradication of such viruses as soon as they appear.